Biologics have been widely used as human therapeutics. Many biologics are produced as recombinant proteins in cell cultures. Charge variants may occur in such recombinant proteins due to post-translational modifications, among others. Charge variants of a recombinant protein may affect the stability, activity, immunogenicity, and pharmacokinetics of the recombinant protein. More particularly, monoclonal antibodies have a wide range of acidic species variants, including those associated with the addition of covalent adducts as well as the chemical degradation at specific peptide regions on the antibody. These variants play an important role in the overall heterogeneity of recombinant therapeutic proteins and are typically monitored during their manufacturing to ensure they fall within regulatory limits.
Heterogeneity may be caused by different types of post-translational modifications. For review, see Walsh, 2006 and Liu, 2008. Heterogeneity manifested as charge variants is often observed in recombinantly expressed proteins. Charge variants may result from chemical degradations (e.g., oxidation, deamidation, isomerization, fragmentation) or addition reactions (e.g., glycation adducts, or covalent additions) that may occur at various locations on the proteins. The cumulative effect of these molecular events is structural and conformational changes on the protein molecule, which may, in turn, change the isoelectric point (pI) of the protein, and may even affect the protein's function. For instance, acidic species variants are likely to bestow a net negative charge on the protein, or they may remove additional positive charges. For review, see Du, 2012.
Numerous protein variants caused by acidic species have been reported. While some of these variants only have a nominal impact on the affected protein, others have more profound effects on the functions of the protein. It has been postulated that the degree of impact of a particular charge variant is dependent on where on the protein the impact occurred, and the extent to which the protein has been modified. Changes to the Fc region of an antibody may not be as impactful as changes occurring on the Fab region, where target binding occurs (Du, 2012). Charge variants have been shown to affect the in vitro and in vivo binding characteristics of antibodies (Pardridge, 1994, Pardridge, 1996). Asparagine deamidation has been shown to cause a significant decrease in antigen binding (Vlasak, 2009, Huang, 2005). Glycation has been shown to increase the formation of aggregates (Banks, 2009). Moreover, acidic species have been shown to cause a lower FcRn binding response, even though the in vivo PK appeared unaffected (Khawli, 2010). Taken together, the presence of acidic species variants in therapeutic proteins may potentially impact the efficacy and/or function of the affected proteins.
Various studies have been conducted to determine how acidic species variants are dependent upon the local environment in which the proteins are produced, as well as the environment in which they are stored after purification. Abu Absi et al. described that higher cell culture temperature facilitates an increase in the amount of deamidated species in the recombinant protein expressed. See Abu Absi et al. (2010). However, no data have been reported on the effects of cell culture media on the resulting product quality of recombinant antibodies.